The clinician is concerned with detecting the presence of, and quantitatively measuring, a variety of substances via the use of many different analytical techniques. The most commonly used techniques employ absorbtiometry, both at visible and ultraviolet wavelengths, however, emission, flame photometry and radioactivity are also commonly used. A novel technique, thus far relatively unexplored in chemistry, is that employing the phenomenon of luminescence.
Analyses based on the measurement of emitted light offer several distinct advantages over conventionally employed techniques, including high sensitivity, wide linear range, low cost per test, and relatively simple and inexpensive equipment.
It has been predicted that the phenomenon of luminescence, and more particularly chemiluminescence could have a major impact in two main areas of clinical analysis. First, it may have an important role as a replacement for conventional colorimetric or spectrophotometric indicator reactions in assays for substrates of oxidases and dehydrogenases. In this type of assay the sensitivity of the luminescence indicator reaction may be used to quantitate substrates not easily measured by conventional techniques (e.g., prostaglandins and vitamins).
The second major clinical application of luminescence must be in the utilization of luminescent molecules as replacements for radioactive or enzyme labels in immunoassay.
In each of these major clinical application areas, chemiluminescent reactions can provide a means to achieve a high level of analytical sensitivity.
Chemiluminescence may be simply defined as the chemical production of light. In the literature it is often confused with fluorescence. The difference between these two phenomena lies in the source of the energy which promotes molecules to an excited state. In chemiluminescence this source is the non-radiative energy yielded as the result of a chemical reaction. The subsequent decay of molecules from the excited state back to the ground state is accompanied by emission of light, which is called luminescence. In contrast, in fluorescence, incident radiation is the source of the energy which promotes molecules to an excited state.
From an analytical point of view, the types of luminescence that have engendered the most interest are chemiluminescence and bioluminescence. The latter being the name given to a special form of chemiluminescence found in biological systems, in which a catalytic protein increases the efficiency of the luminescent reaction. Bioluminescent reactions such as the enzymatic firefly process, have been very useful analytically and convert chemical energy to light with a quantum efficiency of 88%.
In contrast to bioluminescence with the longevity and efficiency of the firefly, the history of chemiluminescence (hereinafter referred to as CL), especially that occurring in the non-aqueous phase, is remarkably short. The important aqueous CL substances luminol and lucigenin were discovered in 1928 and 1935, respectively. A series of organic soluble CL materials were developed in the early 1960's based upon a study of the luminescent reactions of a number of oxalate compounds. A typical organic system useful for CL was disclosed by Bollyky et al., U.S. Pat. No. 3,597,362, and claimed to exhibit a quantum efficiency of about 23% compared with about 3% for the best known available aqueous systems.
Chemiluminescence has become increasingly attractive for its potential in the clinical laboratory, especially for use in the analysis of a number of biologically associated materials, and its known applications have been the subject of thorough reviews, see for example: Whitehead et al. (1979) Analytical Luminescence: Its potential In The Clinical Laboratory, Clin. Chem., 25, 9 1531-1546; Gorus et al. (1979) Applications Of Bio- And Chemiluminescence In The Clinical Laboratory, Clin. Chem., 25, 4 512-519; Isacsson et al. (1974) Chemiluminescence In Analytical Chemistry, Analytical Chemica Acta, 68, 339-362.
With few exceptions, most published CL clinical analytical applications have made use of the less efficient but well known diacylhydrazides, acridinium salts, pyrogallol, or lophine structures. It is important to appreciate that due to the nature of the chemical decomposition of the above chemiluminescent structures in the presence of hydrogen-peroxide, or generators of H.sub.2 O.sub.2, as compared to that of the oxidation reaction of diaryloxalate structures, the latter has over 20 times the quantum yield of chemiluminescence, although its requirement for hydrogen peroxide is greater than the former.
Hydrogen peroxide, an essential component in many chemiluminescent reactions, has usually been the species selected for use in detecting the analyte of interest. For example, in the determination of glucose-Auses et al. (1975), Chemiluminescent Enzyme Method For Glucose. Analytical Chemistry, 47, No. 2, 244-248 employed the oxidation of glucose in the presence of glucose oxidase as the source of H.sub.2 O.sub.2 which, in turn, was reacted with luminol to produce chemiluminescence in proportion to the initial glucose concentration. A limit of detection of 8.times.10.sup.-9 M peroxide was obtained with this system. Williams et al. (1976), Evaluation Of Peroxyoxalate Chemiluminescence For Determination Of Enzyme Generated Peroxide. Anal. Chem., 48, 7 1003-1006 in a similar reaction concluded the limit of sensitivity of the peroxyoxalate system is an order of magnitude poorer than that of the luminol system.
Therefore, until now the oxalic ester system (oxalate system) was generally thought to have little utility for analytical purposes due to its inefficient conversion of hydrogen peroxide.
In one embodiment the present invention overcomes the deficiency of H.sub.2 O.sub.2 dependence by making use of the large chemiluminescent reservoir of energy in the oxalate system's chemistry. By using a suitable quantity of hydrogen peroxide and oxalate, a vast amount of energy may be generated in a form which is then released as chemiluminescence upon the introduction of a fluorescer.
Thus, the oxalate, acting in a fashion which can be visualized as analogous to a charged chemical battery, releases the stored energy to the fluorescer-conjugate in the same manner as an electrical switch in a circuit releases the energy of a battery to a lamp. This "switch" action causes chemiluminescence and, by incorporating the fluorescer to a detector of the analyte of interest, one can employ the reaction to trigger a detection system both qualitatively and quantitatively related to the analyte to be measured.
It is, therefore, an object of the present invention to provide for a system for the detection of a biological analyte of interest comprising an encapsulated fluorescer material which has been conjugated to an immunological specie specific to the biological analyte of interest, a means of disrupting the capsule containing the fluorescer and an energy source other than electromagnetic radiation which is capable of activating the fluorescer.
A further object of the present invention is to provide for a qualitative method for the detection of a biological analyte of interest comprising:
(a) labeling an immunological specie specific to the analyte of interest with an encapsulated fluorescer material which is biologically compatible with such specie;
(b) contacting the encapsulated fluorescer labeled specie and the biological of interest to form an encapsulated fluorescer labeled specie/biological complex:
(c) separating the fluorescer labeled specie/biological complex;
(d) disrupting the capsule containing the fluorescer label thus freeing it to solution;
(e) contacting the freed fluorescer with an energy source other than electro-magnetic radiation which is capable of activating the fluorescer label; and
(f) determining the presence or absence of chemiluminescent light emitted from the activated fluorescer.
A further object of the present invention is to provide for a quantitative method for measuring the amount of a biological analyte of interest comprising:
(a) labeling an immunological specie specific to the analyte of interest with an encapsulated fluorescer material which is biologically compatible with such specie;
(c) contacting the encapsulated fluorescer labeled specie and the biological of interest to form an encapsulated fluorescer labeled specie/biological complex;
(d) disrupting the capsule containing the fluorescer label thus freeing it to solution;
(e) contacting the freed fluorescer with an energy source other than electro-magnetic radiation which is capable of activating the fluorescer label; and
(f) determining the presence or absence of chemiluminescent light emitted from the activated fluorescer.
A further object of the present invention is to provide for a novel class of micro encapsulated fluorescer materials which may be conjugated to an immunological specie specific to a biological analyte of interest to provide a means for the detection of such biological.
A further object of the present invention is to provide for a novel class of conjugated microencapsulated fluorescer/biological compositions useful in the detection of various biological analytes of interest.
A further object of the present invention is to provide for test kits for the detection of a biological analyte of interest employing the microencapsulated fluorescer materials described herein.